In general, genetic engineering techniques have been directed to modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven more intransigent than other eukaryotic cells, due not only to a lack of suitable vector systems but also as a result of the different goals involved. For many applications, it is desirable to be able to control gene expression at a particular stage in the growth of a plant or in a particular plant part. For this purpose, regulatory sequences are required which afford the desired initiation of transcription in the appropriate cell types and/or at the appropriate time in the plant's development without having serious detrimental effects on plant development and productivity. It is therefore of interest to be able to isolate sequences which can be used to provide the desired regulation of transcription in a plant cell during the growing cycle of the host plant.
One aspect of this interest is the ability to change the phenotype of particular cell types, such as differentiated epidermal cells that originate in fiber tissue, i.e. cotton fiber cells, so as to provide for altered or improved aspects of the mature cell type. Cotton is a plant of great commercial significance. In addition to the use of cotton fiber in the production of textiles, other uses of cotton include food preparation with cotton seed oil and animal feed derived from cotton seed husks.
Despite the importance of cotton as a crop, the breeding and genetic engineering of cotton fiber phenotypes has taken place at a relatively slow rate because of the absence of reliable promoters for use in selectively effecting changes in the phenotype of the fiber. In order to effect the desired phenotypic changes, transcription initiation regions capable of initiating transcription in fiber cells during development are desired. Thus, an important goal of cotton bioengineering research is the acquisition of a reliable promoter which would permit expression of a protein selectively in cotton fiber to affect such qualities as fiber strength, length or color.
Relevant Literature
Cotton fiber-specific promoters are discussed in PCT publications WO 94/12014 and WO 95/08914, and John and Crow, Proc. Natl. Acad. Sci. USA 89:5769-5773, 1992. cDNA clones that are preferentially expressed in cotton fiber have been isolated. One of the clones isolated corresponds to mRNA and protein that are highest during the late primary cell wall and early secondary cell wall synthesis stages. John and Crow, supra.
U.S. Pat. No. 5,175,095 describes tomato transcriptional factors which can be used to direct the transciption of DNA in ovary tissue. The factors are expressed immediately prior to anthesis and through flowering.
A class of fruit-specific promoters expressed at or during anthesis through fruit development, at least until the beginning of ripening, is discussed in European Application 88.906296.4, the disclosure of which is hereby incorporated by reference. cDNA clones from tomato displaying differential expression during fruit development have been isolated and characterized (Mansson et al., Mol. Gen. Genet. (1985) 200:356-361. Slater et al., Plant Mol. Biol. (1985) 5:137-147). These studies have focused primarily on mRNAs which accumulate during fruit ripening. One of the proteins encoded-by the ripening-specific cDNAs has been identified as polygalacturonase (Slater et al., Plant Mol. Biol. (1985) 5:137-147).
A cDNA clone which encodes tomato polygalacturonase has been sequenced (Grierson et al., Nucleic Acids Research (1986) 14:8395-8603). Improvements in aspects of tomato fruit storage and handling through transcriptional manipulation of expression of the polygalacturonase gene have been reported (Sheehy et al., Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809; Smith et al., Nature (1988) 334: 724-726).
Mature plastid mRNA for psbA (one of the components of photosystem II) reaches its highest level late in fruit development, whereas after the onset of ripening, plastid mRNAs for other components of photosystem I and II decline to nondetectable levels in chromoplasts (Piechulla et al., Plant Molec. Biol. (1986) 7:367-376). Recently, cDNA clones representing genes apparently involved in tomato pollen (McCormick et al., Tomato Biotechnology (1987) Alan R. Liss, Inc., NY) and pistil (Gasser et al., Plant Cell (1989), 1:15-24) interactions have also been isolated and characterized.
Other studies have focused on genes inducibly regulated, e.g. genes encoding serine proteinase inhibitors, which are expressed in response to wounding in tomato (Graham et al., J. Biol. Chem. (1985) 260:6555-6560: Graham et al., J. Biol. Chem. (1985) 260:6561-6554) and on mRNAs correlated with ethylene synthesis in ripening fruit and leaves after wounding (Smith et al., Planta (1986) 168: 94-100). Accumulation of a metallocarboxypeptidase inhibitor protein has been reported in leaves of wounded potato plants (Graham et al., Biochem & BioPhys. Res Comm. (1981) 101: 1164-1170).
Genes which are expressed preferentially in plant seed tissues, such as in embryos or seed coats, have also been reported. See, for example, European Patent Application 87306739.1 (published as 0 255 378 on Feb. 3, 1988) and Kridl et al. (Seed Science Research (1991) 1:209-219).
In animals, the ras superfamily is subdivided into the subfamilies ras members of which are involved in controlling cell growth and division, rab/YPT members of which control secretory processes, and rho members of which are involved in control of cytoskeletal organization (Bourne et al., (1991) Nature 349: 117-127), and number of homologous genes have now been identified in plants (for a review, see Terryn et al. (1993) Plant Mol. Biol. 22: 143-152). None have been found for the important ras subfamily, all but one of the genes identified have been members of the rab/YPT1 subfamily, and there is only one recent report of the cloning of a rho gene in pea (Yang and Watson (1993) Proc. Natl. Acad. Sci. USA 90: 8732-8736).
Little work has been done to characterize the functions of these genes in plants, although one recent report has shown that a small G protein from Arabidopsis can functionally complement a mutant form in yeast involved in vesicle trafficking, suggesting a similar function for the plant gene (Bednarek et al., (1994) Plant Physiol 104: 591-596).
In animals, two members of the rho subfamily, called Rac and Rho, have been shown to be involved in the regulation of actin organization (for a review, see Downward, (1992) Nature 359: 273-274).
Rac1 has been shown to mediate growth factor-induced membrane ruffling by influencing microfilament alignment on the plasma membrane (Ridley et al, (1992) Cell 70: 401-410), whereas RhoA regulates the formation of actin stress fibers associated with focal adhesions (Ridley and Hall, (1992) Cell 70: 389-399).
In yeast, the CDC42 gene codes for a rho-type protein which also regulates actin organization involved in the establishment of cell polarity required for the localized deposition of chitin in the bud scar (Adams et al., (1990) J. Cell Biol. 111: 131-143.
Disruption of gene function, either by temperature shifts with a CDC42-temperature-sensitive mutant in yeast (Adams et al., 1990), or by micro-injection into fibroblasts of mutant Rac or Rho proteins exibiting a dominant negative phenotype (Ridley et al., 1992; Ridley and Hall, 1992), leads to disorganization of the actin network.
In plants, control of cytoskeletal organization is poorly understood in spite of its importance for the regulation of patterns of cell division, expansion, and subsequent deposition of secondary cell wall polymers. The cotton fiber represents an excellent system for studying cytoskeletal organization. Cotton fibers are single cells in which cell elongation and secondary wall deposition can be studied as distinct events. These fibers develop synchronously within the boll following anthesis, and each fiber cell elongates for about 3 weeks, depositing a thin primary wall (Meinert and Delmer, (1984) Plant Physiol. 59: 1088-1097; Basra and Malik, (1984) Int. Rev. of Cytol. 89: 65-113). At the time of transition to secondary wall cellulose synthesis, the fiber cells undergo a synchronous shift in the pattern of cortical microtubule and cell wall microfibril alignment, events which may be regulated upstream by the organization of actin (Seagull, (1990) Protoplasma 159: 44-59; and (1992) In: Proceedings of the Cotton Fiber Cellulose Conference, National Cotton Council of America, Memphis, Tenn., pp 171-192.
Agrobacterium-mediated cotton transformation is described in Umbeck, U.S. Pat. Nos. 5,004,863 and 5,159,135 and cotton transformation by particle bombardment is reported in WO 92/15675, published Sep. 17, 1992. Transformation of Brassica has been described by Radke et al. Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992) 11:499-505.